Polymers containing antifibrotic agents, compositions containing such polymers, and methods of preparation and use

ABSTRACT

The present invention concerns a method for treating fibrotic conditions by administration of an effective amount of an antifibrotic agent. The antifibrotic agent is preferably a proline analog, such as cis-4-hydroxy-L-proline (cHyp). The antifibrotic agent is operatively linked to a monomer or a polymer, with or without a linking compound, e.g., lysine. Intravenous administration is preferred. The present method facilitates the delivery and release of the antifibrotic agent to inhibit collagen accumulation and thereby to treat fibrosis where collegen metabolism is implicated. A reduced quantity of the antifibrotic agent and a corresponding reduction in the potential for toxicity resulting from prolonged administration thereof may be realized.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.07/864,361 filed on Apr. 06, 1992 which was a continuation of U.S.application Ser. No. 07/523,232 filed on May 14, 1990, now abandoned,and a continuation in part of U.S. application Ser. No. 07/726,301 filedJul. 05, 1991, now U.S. Pat. No. 5,219,564, which was a continuation ofU.S. application Ser. No. 07/549,494 filed on Jul. 06, 1990, nowabandoned.

The present invention relates generally to the treatment of fibroticconditions, and to the use of antifibrotic agents for the modificationof such diseases.

The fibrotic conditions addressed herein include changes in thestructure and function of various organs in connection with themetabolism of collagen and other biomolecules. One of the long-termsequelae of hypertension is the deposition of connective tissue in wallsof blood vessels. In hypertensive rates, collagen biosynthesis anddeposition are increased in the aorta, and these effects are reversedwhen blood pressure is lowered by antihypertensive drugs. Treatment ofanimals with experimental hypertension using agents that selectivelyinhibit collagen formation and reduce vascular collagen content,suggests that increased collagen contributes to the maintenance ofhypertension. Although the use of antifibrotic agents has increased theunderstanding of the role of collagen in hypertension and vasculardisease, their application as potential therapeutic agents for chronicconditions has been limited.

Collagen is the most abundant protein in vertebrates. The biosynthesisof collagen involves unique post-translational modification of pro-alphachains. Hydroxylation of prolyl and lysyl residues, a key part ofcollagen formation, is vital for normal triple-helix formation andintermolecular cross-linking. When post-translational processing isinhibited, non-helical procollagen forms which is degraded byintracellular proteases and is secreted into the extracellular matrix ata slower rate as a nonfunctional protein. The incorporation of prolineanalogues, e.g., cis-4-hydroxy-L-proline (cHyp), into nascent pro-alphachains reduces the extracellular accumulation of collagen.

The agents described herein are believed to act more generally byinhibiting collagen synthesis and thereby averting certain of thepathophysiological sequelae of fibrosis, such as atherosclerosis andhypertension. Without limiting the invention to a particular mechanismof action, through the distortion of bond angles and from sterichinderance among polypeptide chains, cHyp inhibits the folding ofpro-alpha chains into a stable triple helix. Other proline analoguessuch as cis-4-fluoroproline, cis-4-bromoproline, 3,4-dehydroproline andazetidine-2-carboxylic acid have similar effects, and can also inhibitother post-translational steps. The compounds 3,4-dehydroproline andazetidine-2-carboxylic acid are examples of proline analogues havingsimilar effects, which can also inhibit other post-translational steps.3,4-dehydroproline inhibits prolyl hydroxylase activity. This prolineanalogue has been administered to humans with pulmonary fibrosis inadult respiratory distress.

The antifibrotic agents described herein are most effective in tissuesundergoing rapid rates of collagen synthesis. For example, collagencomprises about one-third of the dry weight of pulmonary arteries inwhich synthesis increases rapidly following induction of hypertension.Exposure to hypoxia causes constriction of small pulmonary arteries andhypertension develops from sustained vasoconstriction and structuralchanges in the vascular wall. Proliferation of vascular smooth musclecells and connective tissue accumulation thicken the vessel walls andnarrow the lumen of pulmonary arteries. These structural changes causeor contribute to hypertension.

Collagen metabolism has been implicated as a negative factor in otherdiseases and conditions as well. For example, scar tissue is comprisedlargely of collagen. While some scar tissue deposition is expected as aresult of the closure and healing of wounds, excess scar tissue andcollagen based adhesions are often undersirable and unhealthy. Severalproline analogues have been shown to be effective in inhibiting scarformation.

The present invention in particular relates to monomers and polymerswhich contain the antifibrotic compounds described herein,pharmaceutical compositions containing such compounds and variousmethods of preparation and use. In these compounds, cis-hydroxyproline(cHYP) or another antifibrotic agent is the pharmacologically activeagent, useful in controlling the proliferation of collagen or the othertissue changes described herein in detail. This is particularlyimportant in diseases and conditions where collagen is deposited orsynthesized in abnormally high levels, or where collagen is not properlybroken down or removed, contributing to the pathology of the particulardisease or condition.

Unfortunately, it is recognized that cHYP can be potentially toxic ifused improperly, particularly in chronic use. Thus, this drug has hadlimited clinical utility.

The present invention seeks to overcome these disadvantages. One objectof the present invention is to facilitate the use of antifibrotic agentsin the treatment of diseases and conditions in which collagen metabolismis to be modified, such as when excess collagen synthesis or depositionoccurs.

Another object of the present invention is to combine the antifibroticagents described herein with other compounds, e.g., polymers, to improvethe pharmacokinetic profile of these drugs.

Another Object of the present invention is to combine the therapeuticagents with compounds which have little if any toxicity or side effectsof their own.

Another object of the present invention is to enhance the delivery ofthe antifibrotic agents to the site of activity.

Another object of the present invention is to provide antifibroticagents in a variety of polymeric and monomeric forms which can be usedto modify the pharmacokinetic profile or deliver the agent in question.

These and other objects will be apparent to those of ordinary skill inthe art from the teachings herein.

RELATED PUBLICATIONS

The following publications may be of interest to those skilled in theart. These publications are hereby incorporated by reference:

Poiani, G., et al. Amino Acids: Chem. Biol. & Med. Lubec, G. andRosenthal, G. A. (eds) 634-642 (1990).

Poiani, G., et al. J. Appl. Physiol. 68: 1542-(1990).

Kohn, J., et al. J. Am. Chem. Soc. 109: 817-(1987).

Papaioannu, D., et al. Acta Chem. Scand. 44: 243-(1990).

Bowers-Nemia, M. M., et al. Heterocycles 20(5): 817-(1983).

Abuchowski, A., et al. J. Biol. Chem. 252(11): 3578-(1977).

Ajisaka, K., et al. Blochem. Biophys. Res. Commun. 97(3): 1076-(1980).

Ouchi, T., et al. J. Macromol. Sci. - Chem. A24(9): 1011-(1987).

Zalipsky, S., et al. Eur. Polym. J. 19(12): 1177-(1983).

Zalipsky, S., et al. In Polymeric Drugs and Drug Delivery Systems, Dunn,R. L. and Ottenbrite, R. M., eds. Am. Chem. Soc. 469: 91 (1991).

Nathan, A., et al. J. Polym. Preprints 1990 31(2): 213-(1990).

Ertel, S. I., et al. In Polym. Mat. Sol. Eng, American Chem. Soc. 66:486-(1992).

Additionally, each of the priority applications is hereby incorporatedby reference.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, a polymericantifibrotic agent is disclosed which is comprised of a polymericbackbone and an antifibrotic agent, combined such that the antifibroticagent is attached to, complexed with or incorporated into the polymericchain. The polymer can thus be comprised of one or more monomers,prepolymers or antifibrotic agents, and optionally an antifibroticagent-linking compound. The linking compound is typically an amino acidor a short chain peptide which contains more than one reactive group, ora short chain polyfunctional alkyl compound.

Alternatively, the polymer may be synthesized such that the backbone mayincorporate the antifibrotic agent, with or without the linkingcompound. The antifibrotic agent forms a part of the backbone. In thisaspect of the invention, the polymer backbone can be formed from themonomers noted below and the antifibrotic agent.

In accordance with another aspect of the invention, the antifibroticagent can be operatively linked to a monomer, which may optionallyinclude the linking compound, thus forming a monomeric complex which iscomprised of the monomer and the antifibrotic agent optionally linked tothe monomer through a linking compound. This embodiment would thus havea low molecular weight relative to the embodiments described above.

The polymeric and monomeric compounds noted above can be included in apharmaceutical composition in combination with a pharmaceuticallyacceptable carrier.

The pharmaceutically acceptable carrier may be any of those commonlyrecognized vehicles used in the formulation of pharmaceutical products.

Another aspect of the invention involves a pharmaceutical composition asdescribed above, wherein the polymer or monomeric unit is used in thepharmaceutically acceptable carrier, and thus serves as a carriermolecule for delivery of the active compound, and as a component in thedelivery vehicle. A preferred example of this is in the form of aliposome.

The invention also encompasses a method of treatment of diseases orconditions wherein abnormal collagen metabolism is of concern,comprising administering to a mammalian patient in need of suchtreatment at least one of the antifibrotic agents described herein inpolymeric or monomeric form in an amount effective for treating theabnormality in collagen metabolism.

The diseases and conditions in which the antifibrotic agents describedherein are particularly useful include pulmonary conditions, such aspulmonary fibrosis, atherosclerotic conditions, such asarteriosclerosis, renal disorders, such as renal hypertension, hepaticdisorders, such as cirrhosis, skin conditions, such as scarring andwrinkling and like conditions.

The invention described herein further includes a process for producingthe polymeric or monomeric antifibrotic agents described hereincomprising reacting the antifibrotic agent or agents of choice with themonomer or polymer backbone, or with an antifibrotic agent-linkingcompound, under conditions which do not substantially reduce thepharmacological activity of the antifibrotic agent.

Alternatively, the polymer can be formed, with or without theantifibrotic agent-linking compound, and then the antifibrotic agentcondensed with the reactive groups present thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1e represent graphs depicting the effect of a singleintravenous injection of CHyp entrapped in liposomes on rats exposed tohypoxia (10% O₂) for 7 days.

FIGS. 2a-2e represent graphs depicting the effect of reticuloendothelialblockade with empty liposomes on rats exposed to hypoxia (10% O₂) for 7days.

FIG. 3 is a graph depicting endothelial cell uptake of [¹⁴ C]-L-prolineentrapped in liposomes. Data points, mean; bracket, ±SE; n=4. Time, timeof study; percent, percent uptake of radiolabelled liposomes by culturedpulmonary artery endothelial cells.

FIG. 4 shows the localization of fluorescent dye entrapped in liposomesin cultured pulmonary artery endothelial cells. Diffuse uptake offluorescent dye by endothelial cells. Inset, fluorescence of cells withempty liposomes.

FIG. 5 is a graph depicting the uptake of [¹⁴ C]-L-proline in liposomesin selected organs. Percent of total injected dose of [¹⁴ C]-L-proline(ordinate) versus time after injection (abscissa). Data points, mean;bracket, ±SE, n=4.

FIG. 6 is a graph of smooth muscle cell proliferation in the presence ofpolymeric polyethylene glycol (MW 2000) lysine chemically reacted withcHYP via ester linkages.

FIG. 7 is a graph of smooth muscle cell proliferation in the presence ofpolymeric polyethylene glycol (MW 2000) lysine chemically reacted withcHYP via amide linkages.

FIG. 8 is a comparison of the smooth muscle proliferation in thepresence of ester linked cHYP and amide linked cHYP. Both polymericforms are compared to PEG lysine and free cHYP, free cHYP and free tHYP,which is without substantial biological activity, and

FIG. 9 is a graph of cell proliferation using rat lung fibroblasts inthe presence of polyethylene glycol-lysine linked to cHYP via esterlinkages.

DETAILED DESCRIPTION OF THE INVENTION

The description contained herein includes numerous terms which areunderstood by those of ordinary skill, taking into account the followingdefinitions:

The term "antifibrotic agent" refers to chemical compounds which haveantifibrotic activity in mammals. This takes into account the abnormalformation of fibrous connective tissue, which is typically comprised ofcollagen to a greater or lesser degree. These compounds may havedifferent mechanisms of action, some reducing the formation of collagenor another protein, others enhancing the metabolism or removal ofcollagen in the affected area of the body. All such compounds havingactivity in the reduction of the presence of fibrotic tissue areincluded herein, without regard to the particular mechanism of action bywhich each such drug functions.

It is recognized that certain drugs have been used in the treatment ofdiseases or conditions which typically accompany fibrotic changes intissue, such as in the lungs. These overall conditions may be thesubject of distinct treatment modalities for sequellae other than thefibrotic changes which are described herein. For example, in the patientwith pulmonary fibrosis and pulmonary hypertension, such patients may betreated for the fibrotic changes in the lungs independently from anyother treatment which may be rendered for the hypertensive aspects ofthe overall disease.

The term "backbone" is used to describe the portion of the polymersdescribed herein formed by the polymerization of monomeric units andwhich typically forms the structural component of the polymer. Thebackbone may have one or more side chains attached to it. Both thebackbone and the side chains may have functional or reactive groupscontained therein or attached thereto. Some polymers described hereininclude the antifibrotic agent in the backbone, and many of the polymersdescribed herein contain the antifibrotic agent linking compound in thepolymer backbone. In certain polymers, particularly branched polymers,there may be little or no difference structurally between the backboneand the side chains, and the distinction between the two may be lesssignificant. In other polymers, there may be a great difference betweenthese portions of the polymer in reactivity, structure and thebiological properties attributable thereto.

The term "molecular weight" refers to both number average and weightaverage molecular weights when used to describe the polymers of theinvention. When used to refer to monomers, the antifibrotic agent or theantifibrotic agent-linking compound, the term is used in theconventional sense.

The term "linking compound" is not limited to molecules per se, andrefers to compounds, molecules and molecular fragments, e.g., peptides,which can react with the polymer, monomers and antifibrotic agents toattach the antifibrotic agents to the polymer or to incorporate theantifibrotic agents into the polymer. As such, the linking moleculeincludes compounds and the like with more than one reactive group,preferably two or three reactive groups.

The term "reactive group" refers to chemical moieties which are attachedto the polymer or bonds between atoms in the polymer which participatein the chemical reaction between the components involved, e.g., theantifibrotic agent or the linking compound. Examples of reactive groupsinclude without limitation hydroxyl, carboxyl, amine, amide,carbon-carbon double and triple bonds, epoxy groups, halogen or otherleaving groups and the like.

The term "pharmaceutically acceptable carrier" refers to thosecomponents in the particular dosage form employed which are consideredinert and are typically employed in the pharmaceutical arts to formulatea dosage form containing a particular active compound. This may includewithout limitation solids, liquids and gases, used to formulate theparticular pharmaceutical product. Examples of carriers includediluents, flavoring agents, solubilizers, lubricants, suspending agents,binders or tablet disintegrating agents, encapsulating materials,penetration enhancers, solvents, emolients, thickeners, dispersants,sustained release forms, such as matrices, transdermal deliverycomponents, buffers, stabilizers, preservatives and the like. Each ofthese terms is understood by those of ordinary skill.

When desired, the compounds and compositions of the invention may alsoutilize liposome technology to facilitate delivery of the medication tothe desired site. In this case, the liposome may be viewed as part ofthe pharmaceutically acceptable carrier. The liposomes may or may notutilize the polymer described herein in the structure thereof. Hence, ifthe polymer forms part of the liposome, the polymer may also beconsidered part of the pharmaceutically acceptable carrier itself. Ifthe liposome is comprised of components other than the polymer mentionedabove which is linked to or contains the antifibrotic agent, theliposome for purposes of explanation would be considered part of thecarrier and the polymer with the antifibrotic agent attached theretowould be treated as the active compound.

Liposomes have been used to deliver drugs locally in concentrated form.For example, liposomes have been used to deliver cHyp intravenously torats in order to treat experimental pulmonary hypertension and fibrosis.The blood vessels in rats made hypertensive undergo thickening, due inpart to the accumulation of collagen in the vessel walls. The thickeningand stiffening of these blood vessels contribute to increased resistanceto blood flow and ultimately to elevated blood pressure.

The antifibrotic agent may be selected from the group consisting ofL-azetidine-2-carboxylic acid; cis-4-hydroxy-L-proline;3,4-dehydro-L-proline; cis-4-fluoro-L-proline; cis-4-chloro-L-proline;laevo and cis isomers of compounds of the general structural formula:##STR1## wherein R is OH, Cl, F, NH₂, CH₃, OC(O)CH₃, OC-(O)CH₂ CH₃, SH,SCH₃, OCH₃, ONO₂, OSO₂, OSO₃ H, H₂ PO₄, or COOH; L-pipecolic acid;1,2,3,6-tetrahydro-L-picolinic acid; 1,2,3,4-tetrahydro-L-picolinicacid; 1,4,5,6-tetrahydro-L-picolinic acid;1,2,5,6-tetrahydro-L-picolinic acid; 1,2-dihydro-L-picolinic acid; laevoisomers of the compound of the general structural formula: ##STR2##where X is N, S or O; or mixture thereof and a pharmaceuticallyacceptable carrier therefor.

The preferred antifibrotic compounds include cHYP and its analogs. Themost preferred antifibrotic compound is cHYP.

The antifibrotic agents can be operatively linked to the polymer orincorporated into the polymer, to effectuate release therof over time asthe polymer is metabolized. Operatively linked means joined to thepolymer via one or more covalent bonds or conbined with the polymer andphysically associated therewith without the formation of covalent bonds,such as through ionic attraction or through hydrogen bonding.

The polymers which can be included herein are biocompatible polymershaving little or no pharmacologic activity on their own. The polymers,monomers and linking compounds are described in detail in copendingapplication no. 726,301 which has been incorporated by reference above.

Briefly, the monomers which are useful herein include any functionalunits which can be covalently bound to the antifibrotic agent, orpolymerized to form the backbone of the compounds described herein whichcan be operatively linked to the antifibrotic agent. For example,preferred monomers include ethylene and ethylene glycol monomers,certain vinylic or polyphenolic type monomers, povidone and povidonederivatives, monosaccharides, and other monomers which have low levelsof toxicity and little or no pharmacological activity in and ofthemselves. The preferred monomers include ethylene glycol, povidone andcertain monosaccharides, since these can be reacted with theantifibrotic agent with or without the linking molecule and havedesirable solubility characteristics.

Suitable polymers which can be included herein are polymers comprised inwhole or in part of the monomers referred to above. These are referredto in great detail in the copending application. As such, these maypoly(oxyalkylene) polyacids, block copolymers of such polyacids withpoly(amino acids), polyesters and other types of polymers.

The preferred polymers for use herein are polyalkylene oxides, and inparticular, polyethylene and polypropylene glycols which arecopolymerized with amino acids or peptide sequences, which can providependant functional groups, at regular intervals, for antifibrotic agentattachment or crosslinking.

The preferred poly(alkylene oxides) suitable for use herein include thepolymers of polyethylene glycol (PEG), polypropylene glycol,poly(isopropylene glycol), polybutylene glycol, poly(isobutylene) glycoland copolymers thereof. Hence, the backbone of the polymer typicallycontains straight or branched chain alkyl groups of up to four carbonatoms, with up to about 100 repeating units, with the preferred polymercontaining about 10 to 100 repeating units.

The molecular weight of the polymer is not critical, and would dependmainly upon the end use contemplated. In general, the useful numberaverage molecular weight is between about 1,000 and 200,000 daltons, andpreferably about 2000 to about 50,000 daltons. Preferably the polymersused herein are hydrolytically stable; in this case, lower molecularweight polymers can be used.

The most preferred polymers and copolymers included herein are the PEGsand PEGs copolymerized with amino acids or peptides having multiplefunctional groups.

The preferred linking compounds used herein are amino acids and peptideswhich typically contain saturated or unsaturated straight or branchedalkyl groups of up to about six carbon atoms, or alkylphenyl groups, thealkyl portion of which may be covalently bonded to an amine or otherfunctional moiety. The amino acids and peptides containing a low numberof amino acids contained therein, e.g., up to about five, preferably arealpha amino acids, which are naturally occuring. The most preferredamino acids are those containing multiple functional groups. The mostpreferred amino acids are lysine, arginine and cHyp.

Preferred peptides are those which can react with PEG or another polymerand bond via amide, ester or urethane linkages.

To conduct the polymerization reactions referred to above, one canemploy various aspects of polymer chemistry to obtain polymers withlittle variation in the structure or physical parameters. One example ofa polymerization technique which can be used to synthesize the polymersnoted above is an interfacial polymerization between a waterimmiscibleorganic solution containing one or more activated poly(alkylene) oxides,and a water miscible phase containing one or more amino acids orpeptides, having the appropriately protected C-terminals. The aqueoussolution is buffered as appropriate, e.g., to a pH of about 8.0, and theorganic phase is added. After reaction, the mixture can be acidified andseparated, with the organic phase containing the polymer.

It is also possible to form copolymers noted above using numerousalternative methods and reagents which are well understood.

By selecting the appropriate starting materials, one can form a polymerhaving free hydroxyl, carboxyl or amino groups which are reactive withthe reactive groups present in the antifibrotic agents or with thereactive groups of the linking compounds. For example, when the polymerhas pendant carboxyl groups, the antifibrotic agent may be directlyconjugated with the carboxyl group via a hydroxyl or amino group. Aprotection/deprotection reaction scheme can be utilized to block thedesired antifibrotic agent reactive groups when multiple functionalreactive groups are present may react with the same reagent, and allowthe formation of more stable bonds; deprotection can then be undertaken.LIkewise, one or more functional groups preent on the polymer can beprotected.

Using the teachings above, the antifibrotic compounds other than cHypcan be conjugated with the monomers or incorporated into the polymers ofthe invention in a similar manner.

When the polymer selected does not contain the linking molecule in thebackbone, and it contains pendant reactive groups, e.g., carboxylgroups, or if it is otherwise desired, the polymer can be reacted withthe linking compound prior to reaction with the antifibrotic agent. Forexample, pendant carboxyl groups can be reacted with the linkingcompound, e.g., an alkanolamine, under conditions which favor theformation of either ester or amide bonds between these two compounds,after which the antifibrotic agent is added. The reaction between thepolymer carboxyl groups and the linking compound can be conducted in theappropriate solvent and at the appropriate pH to favor the desiredfunctional group formation. After this reaction, if not already in anorganic solvent, the components can be transferred to an organic mediumand a coupling reagent can be added, e.g., dicyclohexylcarbodiimide(DCC) with any appropriate acylating catalyst to conjugate theantifibrotic agent and the polymer.

The above order of reaction can also be reversed; the drug and thelinking molecule are reacted, and then this reaction product is combinedwith the polymer under appropriate reaction conditions.

Another process for conjugating the polymer and the antifibrotic agentinvolves the reaction of pendant reactive groups with a compound havingaidehyde, ketone or carboxyl groups. The polymer can be combined with acompound which forms acyl hydrazino groups, e.g, hydrazine, and theresulting acyl hydrazino moiety can be linked to the aidehyde, ketone orcarboxyl groups, thus forming a hydrazone or diacyl hydrazide linkagebetween the copolymer and the active compound. Hydrazones can be formedwith aldehyde or ketone containing drugs, or by oxidation ofcarbohydrate residues of glycopeptides.

The polymers noted above can optionally be crosslinked to modify theutility thereof, such as to render the compounds less water soluble.Numerous crosslinking agents can be mentioned as useful herein,including diols and higher polyols, polyamines, polycarboxylic acids,polyisocyanates and the like.

If the polymer is crosslinked, it may be desirable to complex theantifibrotic agent with the polymer rather than covalently bond theactive compound to the polymer, either directly or via the linkingcompound, if adequate delivery of the antifibrotic compound can berealized at the site of activity. Thus, non-covalently bound forms arewithin the scope of the invention, since the antifibrotic agent isoperatively linked to the polymer.

It is also desirable to include the monomers described above reactedwith the antifibrotic agent, with or without one or more of the linkingmolecules included. In this aspect of the invention, the antifibroticagent can be reacted directly with the monomer via any of the processesdetailed above. The monomer is substituted for the polymer and reactedwith antifibrotic agent and/or the linking compound. The monomerconjugated with the drug can then be used in the methods of treatmentdescribed below.

The method aspects of the invention involve the administration of apolymer or a monomer as noted above to a patient in need of suchtreatment, in an amount effective to modulate the metabolism ofcollagen, and thus reduce the formation of fibrotic tissue in theeffected area. As mentioned previously, this may entail any of numerousmechanisms of action, such as inhibiting the formation of collagen,enhancing the removal of collagen which is deposited in tissue, orinhibiting the deposition of collagen which would otherwise formfibrotic tissue.

The compounds may be administered in doses ranging from about 0.05mg/kg/day to as high as about 1-2 g/kg/day, by any appropriate route ofadministration, depending upon the particular condition under treatment.The exact dosages will be apparent to those skilled in the medical artstaking into account the teachings contained herein and the overallcondition of the patient. Preferably once-daily dosage will be effectivein treating patients for the disorders described herein, but divideddaily dosages are acceptable as well.

One preferred method of treatment involves the administration of one ormore of the polymeric or monomeric antifibrotic agents described aboveto a mammalian patient with a pulmonary disease or disorder, such aspulmonary hypertension or pulmonary fibrosis. Pulmonary hypertension mayaccompany pulmonary fibrosis in some patients, or may be foundindependent of other pulmonary disease, such as in congestive heartfailure or other hypoxic conditions. In this method of treatment, theantifibrotic agent may be administered in polymeric or monomeric formvia any of the preferred routes of administration, e.g., oral,parenteral, aerosol, e.g., IPPB.

Another preferred method of treatment involves the administration of oneor more of the antifibrotic agents described above to a mammalianpatient with hepatic disease characterized by a defect in collagenmetabolism, e.g, cirrhosis. In this method of treatment, theantifibrotic agent is preferably administered in polymeric or monomericform, via any route of administration, preferably oral or parenteral.

Another preferred method of treatment involves the administration of oneor more of the polymeric or monomeric antifibrotic agents describedabove to a mammalian patient with a skin disorder wherein collagenmetabolism, e.g., excessive deposition is implicated. Examples of suchskin disorders include the excess or abnormal formation of scar tissue,wrinkling, scleroderma and other conditions involving the skin. In thismethod of treatment, the antifibrotic agent is most preferablyadministered orally, parenterally, topically or transdermally.

Another preferred method involves the treatment of non-specific vasculardisease, e.g., atherosclerosis, wherein the polymeric or monomericantifibrotic agent is administered to a mammalian patient withatherosclerotic disease in an amount effective to treat abnormalcollagen deposition or metabolism. Atherosclerotic disease involves theformation of atherosclerotic plaque and other changes in the vasculartissue, such as thickening of the vessel walls, which may involvecollagen to a greater or lesser degree. In this method of treatment, theantifibrotic agent is most preferably administered orally, parenterally,topically or transdermally.

The invention described herein also includes various pharmaceuticaldosage forms containing the antifibrotic agents in polymeric ormonomeric form. The pharmaceutical dosage forms include those recognizedconventionally, e.g., tablets, capsules, oral liquids and solutions,drops, parenteral solutions and suspensions, emulsions, oral powders,inhalable solutions or powders, aerosols, topical solutions,suspensions, emulsions, creams, lotions, ointments, transdermal liquidsand the like.

Typically the dosage forms comprise from about 5 to about 70 percentactive ingredient per dosage unit. These may be packaged in multipledose containers or unit dose packages.

Suitable solid carriers are known, e.g., magnesium carbonate, magnesiumstearate, talc, lactose and the like. These carriers are typically usedin oral tablets and capsules.

Oral liquids may also likely comprise about 5 to about 70 percent activeingredient in solution, suspension or emulsion form. Suitable carriersagain are known, and include, e.g., water, ethanol, propylene glycol andothers.

Aerosol preparations are typically suitable for nasal or oralinhalation, and may be in powder or solution form, in combination with acompressed gas, typically compressed air. Additionally, aerosols may beuseful topically.

Topical preparations useful herein include creams, ointments, solutions,suspensions and the like. These may be formulated to enable one to applythe appropriate dosage topically to the affected area once daily, up to3-4 times daily as appropriate. Topical sprays may be included herein aswell.

Depending upon the particular compound selected, transdermal deliverymay be an option, providing a relatively steady state delivery of themedication which is preferred in some circumstances. Transdermaldelivery typically involves the use of a compound in solution, with analcoholic vehicle, optionally a penetration enhancer, such as asurfactant and other optional ingredients. Matrix and reservoir typetransdermal delivery systems are examples of suitable transdermalsystems. Transdermal delivery differs from conventional topicaltreatment in that the dosage form delivers a systemic dose of medicationto the patient.

A delivery system which may have particular utility in the presentinvention is one which utilizes liposomes to encapsulate or include theantifibrotic agent. In this system, the liposome may be targeted to aparticular site for release of the antifibrotic agent or degradation ofthe polymeric or monomeric structure to release the active compound.This delivery system thus may obviate excessive dosages which are oftennecessary to provide a therapeutically useful dose of the drug at thesite of activity. In selected experiments, and as set forth in theexamples, the effective amount of the antifibrotic agent may be reducedby as much as twenty times the normal effective dose, as indicated byexperimental protocols wherein the same antifibrotic agents areadministered in free form.

Liposomes may be used herein in any of the appropriate routes ofadministration described above. For example, liposomes may be formulatedwhich can be administered orally, parenterally, transdermally or viainhalation. Drug toxicity could thus be reduced by selective drugdelivery to the effected site using liposomes. If, for example the drugis liposome encapsulated, and is injected intravenously, the liposomesused are taken up by vascular cells, and locally high concentrations ofthe drug could be released over time within the blood vessel wall,resulting in improved drug action.

The use of liposome encapsulated polymeric and monomeric antifibroticagents finds utility in the treatment of pulmonary hypertension, and itsassociated events and sequelae, such as, for example, polycythemia.Liposome encapsulation permits greater quantities of the effective agentto be administered without concomitant toxicity and thereby offers aviable therapeutic alternative.

The liposome encapsulated materials are preferably administeredparenterally and, particularly may be administered by intravenousinjection. A particularly preferred proline analog iscis-4-hydroxy-L-proline. The proline analogs of the present inventionare generally disclosed in U.S. Pat. No. 4,428,939, issued Jan. 31, 1984to Darwin J. Prockop, the disclosure of which is incorporated herein byreference. Such compounds are illustrative of antifibrotic agents usefulin accordance with the present invention.

It has been demonstrated that twice daily subcutaneous injections of 200mg/kg cHyp ameliorates development of chronic hypoxia-inducedhypertension in rats. Since prolonged treatment with cHyp causestoxicity in adult rodents, localized delivery of cHyp to hypertensivepulmonary arteries has been achieved by encapsulation in phospholipidbased liposomes. Rats with experimentally induced pulmonary hypertensionhave been successfully treated with liposome-encapsulated cHyp, reducingthe effective dose of drug substantially, and causing sustainedinhibition of vascular collagen accumulation.

The invention can be further illustrated in connection with thefollowing Examples. For purposes of illustration, when a PEG copolymeris reacted with lysine, the following poly(PEG-Lys) copolymer may beformed: ##STR3## Likewise for purposes of illustration, the followingreaction schemes are included within the preferred processes of makingthe cHyp based polymers of the present invention. Scheme 1 involves thepreparation of poly(cis-N-palmitate-Hyp) ester and is the subject ofExample 13 below. The trans-N-palmitoyl hydroxyproline is reacted withtriphenylphosphine and a dehydrating agent to form a bicyclic compound,which in turn opens and rearranges to the cis form, which can bepolymerized. Scheme 2 involves the preparation of monomethoxy-PEG-cHYPconjugates, and is described in detail in Example 14. Scheme 3illustrates the preparation of poly(PEG-Lys)-cHyp copolymers, and isdescribed in detail in Example 15. ##STR4##

EXAMPLE 1 PREPARATION OF PEG-BIS SUCCINIMIDYL CARBONATE

The preparation of PEG-bis succinimidyl carbonate is disclosed in U.S.application Ser. No. 340,928. In a 250 mL round bottomed flask, 10 g (10mmols of hydroxyl groups) of PEG 2000 (Fluka) was dissolved in 120 mL oftoluene and the polymer solution was azeotropically dried for two hoursunder reflux, using a Dean-Stark trap. The polymer solution was thencooled to 25 degrees C. and 15 mL (29 mmol) of a 20 percent solution ofphosgene in toluene (1.93 M) was added. The reaction mixture was stirredat 25° C. overnight and then evaporated to dryness on a rotaryevaporator (water bath temperature maintained at 40° C.). Another 100 mLof toluene was added and evaporated to remove all traces of phosgene. Tothe polymeric chloroformate was added 30 mL of dry toluene, 10 mL ofmethylene chloride, and 1.7 g (14.8 mmol) of N-hydroxy succinimide, andthe mixture was stirred vigorously. The reaction flask was then cooledin an ice water bath and 1.5 g (14.9 mmol) of triethylamine was addedgradually. Immediate precipitation of triethylamine hydrochloride wasseen. The cooling bath was removed and the stirring continued at 25degrees C. for five hours. Then 10 mL of toluene was added and thereaction mixture cooled to 4 degrees C. to maximize the triethylaminehydrochloride precipitation.

The precipitate was filtered and the filtrate concentrated to about halfof its original-volume. The concentrated solution was then added to 60mL of ether with stirring to precipitate the polymeric product. Aftercooling to 40 degrees C., the crude product was recovered by filtration,dried, redissolved in 100 mL of 2-propanol at 45 degrees C. and allowedto recrystallize. The product was recovered by filtration, washed withether and dried under high vacuum. The recovery of the white crystal andsolid was 74 percent.

EXAMPLE 2 Preparation of PEG-Lysine Ethyl Ester Copolymer(Poly(PEG-Lys-OEt)

In a 500 mL three-necked round-bottomed flask fitted with an overheadstirrer was dissolved 1.1 g (4.4 mmol) of lysine ether esterhydrochloride salt (Fluka) and 1.7 g (21 mmol) of sodium bicarbonate in100 mL of water. The PEG-N-hydroxy succinimide-dicarbonate of Example I(10 g, 4.4 meq) was dissolved in 200 mL of methylene chloride and addedto the reaction mixture. The mixture was stirred vigorously (about 1100rpm) for two hours and then acidified to about pH 2. The two phases wereseparated and the organic phase was washed twice with NaCl. The organiclayer was then dried over anhydrous MgSO₄, filtered and concentrated.The polymer was precipitated using cold ether, cooled to 40 degrees C.and filtered to recover 6.7 g (67 percent) of the polymer.

The crude polymer (500mg) was dissolved in 10 mL of distilled water anddialyzed against distilled water at room temperature for 48 hours usinga SPECTRAPOR(tm) membrane with a molecular weight cut-off of 12,000 to14,000 daltons. The purified polymer was extracted with methylenechloride, washed with saturated NaCl solution, dried and evaporated toobtain 263 mg (53 percent) of pure polymer.

EXAMPLE 3 Preparation of PEG-Lysine Copolymer Poly(PEG-Lys)

The polymer of Example 2 (5 g) was dissolved in 5 mL of H₂ O. The pH ofthe polymer solution was about 5 as measured with a pH meter. A 0.01 NNaOH solution was prepared, and the base was added dropwise into thepolymer solution with stirring. The pH was monitored continuously andkept around 11.5 by the addition of base as needed. The reaction wasallowed to go for five hours. The reaction was stopped and the reactionmixture was acidified with 0.1 N HCl. The polymer was extracted intomethylene chloride and the extract was washed with saturated Nacl, driedover anhydrous MgSO₄, filtered and concentrated. The polymer was thenprecipitated with cold ether. After cooling for several hours, theproduct was collected in a Buchner funnel, washed with cold ether anddried under vacuum overnight. 3.5 g of polymer (71 percent) wasrecovered.

EXAMPLE 4 Preparation of Activated Poly (PEG-Lys)

In a 10 mL round-bottomed flask, 1.0 g (0.46 mmol) of the polymer ofExample 3 was dissolved in 5 mL of methylene chloride. To this solution,0.26 g of N-hydroxysuccinimide (Aldrich) (2.3 mnol) was added. The flaskwas cooled in an ice water bath and 0.10 g (0.50 mmol) ofdicyclohexylcarbodiimide (DCC) (Aldrich) was added. The reaction mixturewas then stirred at 0 degrees C. for one hour and at room temperatureovernight. The reaction mixture was filtered to remove dicyclohexyl ureaand the methylene chlorine was evaporated to give a white, waxymaterial.

Isopropanol (5 mL) was added and the mixture was stirred until a clearsolution was obtained. Cooling to -15 deg C. precipitated a white solidwhich was collected on a Buchner funnel and washed first withisopropanol and then with hexane. The material was further purified byrecrystallization from isopropanol. The recovery of the final productwas 0.72 g (71 percent).

EXAMPLE 5 Preparation of Poly(PEG-Lys) with Pendant Acyl HydrazineFunctional Groups

In a 50 mL round-bottomed flask, 2.2 g (1.0 mmol) of the polymer ofExample 3 was dissolved in 20 mL of methylene chloride. The flask wasthen cooled in an ice water bath. To the flask were added 410 mg (2.0mmol) of DCC and 260 mg (2.0 mmol) of tert-butyl carbazate (Aldrich).The contents of the flask were stirred at ice water bath temperature for1 hour and then stirred at room temperature for 24 hours. The reactionmixture was filtered to remove the dicyclohexyl urea, followed byevaporation of the filtrate to dryness, which gave 1.5 g of light solidthat was purified by recrystallization from 2-propanol. The ¹ H protonNMR spectrum of the white, waxy solid showed tert-butyl peaks, the areaof which corresponded to greater than 90 percent conversion. Whenredissolved in methanol and reprecipitated with ether, the relativeintensity of this peak did not decrease.

An approximate 4 M solution of HCl in dioxane was prepared by bubblingHCl gas through dioxane in an Erlenmeyer flask (a 4.0 M solution is alsoavailable commercially from (Pierce). In a 250 mL round-bottomed flaskwas placed 75 mL of the 4.0 M HCl/dioxane solution, and to this wasadded with stirring 5.0 g of the polymer-carbazate reaction product inthe form of small pieces. Stirring was continued for two hours at roomtemperature. The polymer settled at the bottom of the flask as an oil.The dioxane/HCl layer was decanted out and the polymer layer was addedto 100 mL of the ether with stirring. The polymer precipitated and wasisolated, washed twice with 50 mL of ether and dried under vacuum. Itwas further precipitate_(d) by recrystallization from isopropanol.

The ¹ H NMR spectrum of the product showed the complete absence oftert-butyl groups. Non-aqueous titration against sodium methoxide withmethyl red as the indicator showed about 100 percent of the expectedhydrochloride.

EXAMPLE 6 Preparation of Poly(PEG-Lys) Having Ethanol Amide PendantFunctional Groups

In a 50 mL round-bottomed flask, 0.400 g (0.1819 mmol) of thepoly(PEG-Lys) of Example 3 was dissolved in 40 mL of water. To thissolution was added 0.1 mL (1.656 mmol) of ethanol amine (Aldrich). ThepH was adjusted to 4.75 by the addition of 0.1 N HCl. Then 0.348 g (1.82mmol) of solid 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) (Sigma) wasadded. The pH had a tendency to increase, but was maintained around 4.75by the addition of 1 N HCl. After 30 minutes, no further increase in pHwas observed. The reaction mixture was stirred overnight and thenacidified and extracted into methylene chloride. The methylene chlorideextract was washed with saturated sodium chloride solution, dried withanhydrous magnesium sulfate, filtered, concentrated to a viscous syrupand precipitated with cold ether. About 0.318 g of crude poly(PEG-Lys)with ethanol amide pendant functional groups was recovered. The crudeproduct was purified by reprecipitation from isopropanol, followed bywashings with hexane and complete drying in vacuo. Thin layerchromatography (TLC) in a 4:1 ratio solution of ethanol to ammoniashowed an absence of free ethanolamine.

EXAMPLE 7 Preparation of Poly(PEG-Lys) Having Ethylamine PendantFunctional Groups

In a 100 mL three-necked flask, 1.21 g (0.55 mmol) of the poly(PEG-Lys)of Example 3 was dissolved in 80 mL of water. To this solution was added0.37 mL (5.5 mmol) of ethylene diamine (Aldrich). The pH was adjusted to4.75 by the addition of 1 N HCl. Then 1.05 g (5.5 mmol) of solid1-(3-dimethylaminopropyl-3-ethylcarbodiimide) was added. The pH had atendency to increase, but was maintained around 4.75 by the addition of1 N HCl. After 30 minutes, no further increase in pH was observed. Thereaction mixture was stirred overnight and then made basic and extractedinto methylene chloride. The methylene chloride extract was washed withsaturated sodium chloride, dried with anhydrous magnesium sulfate,filtered, concentrated to a viscous syrup and precipitated with coldether. About 0.725 g of crude poly(PEG-Lys) having ethylamine pendantfunctional groups was recovered, which was purified by reprecipitationwith isopropanol. TLC in a 2:1 solution of ethanol to ammonia showed anabsence of free diamine.

EXAMPLE 8 Preparation of Poly(PEG-Lys) Having Pendant HexylamineFunctional Groups

The procedure of Example 7 was followed substituting 5.5 mmol ofhexamethylene diamine (Aldrich) for the 5.5 mmol of the ethylenediamine. Upon purification of the product, TLC in a 2:1 ratio ethanol toammonia solution showed an absence of free diamine.

EXAMPLE 9 Preparation of N-Benzylcarbamate Derivative of a Copolymer ofPEG and Glutamic Acid

2 g of PEG 2000 was azeotropically dried following the procedure ofExample 1 by dissolving the polymer in 30 mL of toluene in a preweighed50 mL round-bottomed flask provided with a stirrer. The polymer solutionwas azeotropically dried for two hours under reflux in an oil bath, thetemperature of which was maintained at 140 degrees C. All the solventwas distilled off and the product was dried under vacuo. The dried PEGwas reweighed, dissolved in 5 mL of methylene chloride and stirred underargon. An equimolar amount of glutamic acid, the N-terminal of which wasprotected by a benzylcarbamate functional group (Sigma) was added. Fourtimes this amount of diisopropylcarbodiimide (Aldrich) and four timesthis amount of dimethylaminopyridinium toluene sulfonate (Aldrich) wereadded. The reaction mixture was heated slightly to dissolve the glutamicacid. The reaction was allowed to run for 24 hours at room temperaturewith stirring. A urea precipitate formed that was removed by filtration,and the product was precipitated by cold ether, filtered and dried undervacuum. About 1.6 g of polymer was recovered, which was purified byreprecipitation from isopropanol. TLC in a 5:5:1 ratio solution oftoluene to acetic acid to water showed the absence of free glutamicacid.

EXAMPLE 10 Preparation of Poly(PEG-Lys) Cross-Linked by HexamethyleneDiisocyanate

A mold was prepared by clamping two square glass plates together, one ofwhich had a 5 cm diameter circular cavity. The contacting surfaces ofthe glass plates were coated with trimethylchlorosilane (Aldrich) toprevent adhesion. The mold was placed on a level surface inside a glovebox and further leveled using a carpenter's level. In a 100 mL beaker,1.5 g of the poly(PEG-Lys) having pendant acyl hydrazine groups (0.67mmol of hydrazine groups) of Example 5 was dissolved in 40 mL ofmethylene chloride. To this solution was added 1.5 g of finely powderedsodium bicarbonate. The suspension was stirred for one hour and thesupernatant was tested for the presence of chloride ions with silvernitrate. A few drops of the methylene chloride solution were placed intoa test tube, the methylene chloride was evaporated, ahd the residue wasreacted with a few drops of silver nitrate solution acetified withnitric acid. The absence of any white turbidity indicated the completeneutralization and removal of hydyochloric acid.

The solution was then filtered and the residue was washed with methylenechloride. To the combined filtrate, 54 microliters of hexamethylenediisocyanate (56 mg,, 0.67 meq of isocyanate groups) (Aldrich) was addedwith stirring. After two to three minutes of stirring, the solution waspoured into the circular cavity of the solvent casting mold. The cavityof the mold was covered with filter paper so that the solventevaporation was slow and uniform. The film was allowed to dry in theglove box for 48 hours and then peeled from the mold. The thickness ofthe membrane was measured with an electronic vernier caliper inside theglove box and was found to be about 0.1 mm.

The membranes obtained were semi-transparent and were somewhathygroscopic, curling up when exposed to moisture in ambient air. Whenplaced in water, the size of the films doubled in all dimensions,indicating a very large swelling ratio. The swollen membranes weretransparent. The membrane was assayed with trinitrophenyl sulfonic acid(TNBS) (Fluka) to determine the extent of crosslinking. An excess ofTNBS was used, and after reacting with the polymer, the unreacted TNBSwas allowed to react with an excess of adipic hydrazide. The IRabsorbance obtained at 500 nm was then used to calculate the amount offree hydrazides present on the cross-linked membrane. Using this method,it was found that 80-85 percent of all available hydrazides precipitatedin cross-linking, leaving only 15-20 percent of unreacted hydrazides onthe cross-linked membrane.

Calorimetry of the cross-linked membrane showed a sharp endothermictransition at 33.4 deg C. This is very similar to the T_(m) of thecorresponding non-cross-linked poly(PEG-Lys) having pendant acylhydrazine functional groups (34.1 deg C.). When the membrane was heatedin an oven above the phase transition temperature, it became veryflexible but did not disintegrate. These results indicate that theproperties of PEG dominate even after copolymerization with lysine andcross-linking.

Swelling measurements of the membrane were made by two methods. Thedimensions of the dry membrane were measured and the membrane wasallowed to swell in water. The increase in dimension was taken as ameasure of swelling. Alternatively, the membrane was weighed before andafter swelling and the increase in weight was taken as a measure ofswelling. Both methods indicated that the membrane absorbs about 5 to 8times its weight of water.

The tensile strength of the membrane was measured using strips ofmembrane 0.07 mm thick, 5 mm wide and 50 mm long. Measurements were madeemploying both dry and swollen membranes.

In the swollen state, the membrane behaves like a perfect elastomer. Themembrane did not exhibit a yield point and a plot of stress againststrain gave a straight line.

The stability of the membrane was investigated in acidic, basic andneutral media, the results of which are listed in the Table below. Smallspecimens of the membrane were placed in contact with a number ofaqueous solutions of varying pH at room temperature and the timerequired for the complete disappearance of the membrane was noted. Themembrane was generally found to be more stable in weakly acidic mediaand extremely unstable in alkaline media.

                  TABLE 1                                                         ______________________________________                                                         TIME REQUIRED FOR                                            SOLUTION         DISAPPEARANCE                                                ______________________________________                                        1 N HCL          5 to 8 days                                                  0.1 N HCL        No change in 8 days                                          0.01 N HCL       No change in 8 days                                          Deionized water  No change in 8 days                                          Borate (pH = 9)  5 to 8 days                                                  0.01 N NaOH      Less than 5 hours                                            0.1 N NaOH       Less than 5 hours                                            1 N NaOH         Less than 1 hour                                             ______________________________________                                    

To test the stability under physiological conditions, an acceleratedstability study was performed in which samples of membrane were exposedto phosphate buffer of pH 7.4 at 60 deg C. Under these conditions, themembrane lost weight at the rate of about 1 percent per hour. After 60hours, the membrane disintegrated and became soluble in the buffer.

EXAMPLE 11 Preparation of Poly(PEG-Lys) Membranes Cross-Linked withTris(Aminoethyl) Amine

In a 100 mL beaker, 1.87 g of the PEG bis(succinimidyl carbonate) ofExample 1 was dissolved in 20 ml of methylene chloride. In anotherbeaker, 82 microliters (89 mg) of tris(aminoethylamine) was dissolved in20 ml of methylene chloride. The triamine solution was added to the PEGsolution with vigorous stirring. After about five minutes, films werecast of the solution following the procedure described above withrespect to Example 16.

Swelling measurements of the membrane were made by the two methodsdescribed above with respect to Example 16. Both methods indicated thatthe membrane absorbed about six times its weight of water.

The stability of the membrane was investigated in acidic, basic andneutral media as described above. In sodium hydroxide (0.01 and 0.1 N)the membrane disintegrated within a few hours. In acidic media and inphosphate buffer (pH 7.4) the membrane appeared to be stable for longerperiods of time. The accelerated degradation study of Example 10 wasalso performed, in which the membrane remained intact for more than aweek. An analysis of the buffer in which the accelerated stability studywas conducted revealed that during the first 24 hours a small amount ofPEG chains had leached from: the crosslinked membrane, but throughoutthe following 72 hours, no more PEG was leached.

EXAMPLE 12 Preparation of Poly(Caprolactone) Semi-IPN's of Poly(PEG-Lys)Membranes Cross-Linked by Diisocyanate

The poly(PEG-Lys) membrane cross-linked by diisocyanatohexane wasprepared as in Example 10, using 210 mg of the poly(PEG-Lys) of Example5 having acyl hydrazine functional groups, dissolved in 10 mL ofmethylene chloride. The free base was formed with sodium bicarbonate,and the solution was then filtered. Prior to the addition of fourmicroliters (3.9 mg) of the hexamethylene disocyanate, 0.47 g ofpoly(caprolactone) (Union Carbide) (mw 72,000) was added to thefiltrate, which was stirred for 30 minutes to dissolve the polymercompletely. The poly (PEG-Lys) was cross-linked and films were castfollowing the procedure described above with respect to Example 16. Theresulting membrane was hydrophilic and absorbed water with anequilibrium water content of 36%, whereas films made ofpoly(caprolactone) alone is hydrophobic.

EXAMPLE 13

A. Poly(cis-N-Pal-Hyp ester)

A poly(cis-N-Pal-Hyp) ester was prepared by melt transesterification ofcis-4-hydroxy-N-palmitoyl-L-proline methyl ester (3) in the presence ofaluminum isopropoxide (1% w/w), following a method described in J. Am.Chem. Soc. 109:817 (1987) for the polyesterification of N-protectedtrans-hydroxy-L-proline (Scheme 1). The monomer (3) was prepared fromcis-hydroxy-L-proline(6) by conventional methods or from trans-N-Pal-Hyp(1) by reaction with triphenylphosphine (TPP) and diethylazodicarboxylate (DEAD), via the bicyclic lactone(2) as described byPapaioannu et al. in Acta. Chem. Scand. 44:243 (1990).

B. Poly(ethylene glycol)-cis-Hyp conjugates (12) and (13)

Cis-N-Boc-L-proline methyl ester (10) was esterified with the succinicester of momomethoxy-PEG (8) in presence of DCC/dimethylaminopyridine(DMAP), followed by deprotection of the cis-Hyp-N-terminus with a 4NHCl/dioxane solution to yield the conjugate (12) (Scheme 2a). Conjugate(13) was prepared by reaction of the succinimidyl carbonate activatedmonomethoxy PEG (9) with the lactone (11), followed by hydrolysis of thelactone in 2N KOH (Scheme 2b). Lactone (11) was prepared fromtrans-N-Boc-Hyp as described for compound 2 (Scheme 1), followed bydeprotection of the N-terminus with 4N HCl/dioxane.

C. Poly(PEG-Lys-cis-Hyp) copolymers (16) and (17)

were prepared by covalent attachment of the Hyp derivatives (10) and(11) to the pendant side chains of poly(PEG-Lys) as described for thePEG conjugates (12) and (13) (Scheme 3a and 3b). The extent of cis-Hypattachment to the poly(PEG-Lys) copolymer was assessed by the ratio ofLys to Hyp as determined by amino acid analysis.

D. Polyethylene Qlycol-cHyp conjugated 1:2 Ratio

Polyethylene glycol may be conjugated with cHyp according to thefollowing reaction scheme, resulting in a conjugate containing two cHypmoieties. As shown in the reaction pathway on the left, the cHyphydroxyl groups may be reacted with a conjugate of PEG and succinicacid, thus forming multiple ester linkages. The cHyp carboxylic acidgroup can be protected with a methoxy group or another suitableprotecting group.

In the reaction scheme on the right, the PEG is linked to two cHypmoieties through urethane linkages. ##STR5##

Results

Poly(cis-4-hydroxy-N-palmitoyl-L-proline ester)

Since only the cis isomer of Hyp is pharmacologically active, thepolymerization conditions were analyzed for effects on the retention ofthe cis configuration. The polymerization reaction was performed attemperatures ranging from 180° to 210° C. Polymers of highest molecularweight (Mw=21,600, Mn=15,900) were obtained when the reaction wasconducted at 195° C. for 5 h. All polymers were then hydrolyzed in 1MNaOH and the conformation of Hyp formed during hydrolysis was determinedby ¹³ C NMR.

A comparative hydrolysis of poly(trans-N-Pal-Hyp ester) obtained by thesame method at 180° C. from trans-N-Hyp-Me showed that only trans-Hypwas formed. In contrast, hydrolysis of the polyesters obtained from thecis monomer led to mixtures of cis and trans isomers, which could beresolved due to a chemical shift difference of almost 1 ppm between thepyrrol ring carbons of the two isomers. Comparing the peak heights of ¹³C NMR spectra to a calibration curve obtained from mixtures of knowncompositions, facilitated a quantitative analysis of the hydrolysismixtures (Table A).

                  TABLE 2                                                         ______________________________________                                        EFFECT OF POLYMERIZATION CONDITIONS                                                                            cis/trans                                    T (°C.)                                                                       Time (h) Mw         Mn    ratio                                        ______________________________________                                        180    17       *          *     9/1                                          195     5       21,590     15,856                                                                              3/1                                          210    17       14,224     10,166                                                                              1.8/1                                        210     5       15,644     11,377                                                                              not determined                               ______________________________________                                         *Mw and Mn could not be determined due to the high polydispersity of the      sample                                                                   

Since increasing the reaction temperature favored the undesirableformation of trans-Hyp, reaction conditions were optimized at 180° C.Polymers of very low molecular weight were obtained. At 210° C.,polymers with a low cis/trans ratio were formed. However at 195° C., itwas possible to prepare relatively high polymers which consistedpredominately of cHyp.

Alternatively, a ring opening polymerization reaction can be run usingthe bicyclic lactone (2). The polymerization reaction was performed at140° C. for variable periods of time (15 h to 5 days), using aluminumisopropoxide as the catalyst. This procedure gave low molecular weightpolymers that consisted of an almost equimolar mixture of cis andtrans-N-Pal-Hyp (cis/trans ratio: 0.9/1). Attempts to synthesize thetarget polymer using a coupling agents, such as DCC, in a directcoupling reaction failed due to the formation of the bicyclic lactone(2), via intramolecular esterification.

EXAMPLE 14 ATTACHMENT OF CIS-HYP TO POLY(ETHYLENE GLYCOL) DERIVATIVES

Due to their physicochemical and biological properties, poly(ethyleneglycols) (PEGs) are promising drug carriers. Attachment of PEG toproteins was found to increase blood circulation time of the PET-proteinconjugates and to delay clearance by the RES.

The attachment of cis-Hyp has been attached to two differentpoly(ethylene glycol) based carriers. In the first case, cis-Hyp wasattached to a monomethoxy-PEG (Mw=5,000) unit leading to new cis-Hypconjugates having a 1:1 ratio of PEG to cis-Hyp (Scheme 2a and 2b). In asimilar fashion cis-Hyp was attached to poly(PEG-Lys), a new polymericdrug carrier. In poly(PEG-Lys), PEG chains and L-lysine are connectedvia urethane bonds in a strictly alternating fashion. The carboxylicgroups of the lysyl residue provide convenient anchors for theattachment of the pendant ligands. c-Hyp was bound to the PEG basedcarrier by labile ester bonds (Scheme 3a) and by more stable amide bonds(Scheme 3b).

EXAMPLE 15 LIPOSOME ENCAPSULATION

The encapsulation of the antifibrotic monomers and polymers of thepresent invention into liposomes may proceed in accordance with knowntechniques. An example of the preparation of the liposome encapsulatedproline analogue of the invention follows: Small unilamellar liposomeswere prepared by reverse phase evaporation using the method of Szoka andPapahadjopoulos as modified by Turrens and associates. A stock solutioncontaining 97.5 mg L-alpha-dipalmitoyl lecithin, 24.2 mg cholesterol,and 9.6 mg stearylamine in a 14:7:4 molar ratio was dissolved in 5 ml ofchloroform in a 50 ml round bottom flask. To this mixture, 50 mg of cHypdissolved in 2.5 ml of 10 mM phosphate-buffered saline (PBS), pH 7.4,was added. The mixture was sonicated (model W-385 Ultrasonic Processor,Heat Systems-Ultrasonics, Inc., Farmingdale, N.Y.) at a power output of7 for 1 min at 10° C. The mixture was converted to a homogeneous milkyemulsion which was slightly viscous. The emulsion was transferred to a50 ml rotary evaporation flask and volume was reduced under vacuum (400torr) while maintaining the temperature at 25° C. When the emulsionbecame viscous and did not pool in the flask, 1.25 ml PBS was added. Theevaporation was continued at 49° C. until the odor of chloroform was nolonger detected and a free flowing turbid suspension was present. Thesuspension was kept at 4° C. overnight, centrifuged at 100,000 xg for 35min at 4° C., and recentrifuged after suspending the pellet in 6.5 ml ofPBS. Prior to injection, the pellet was stored at 4° C. in 2.5 ml of PBS(40 μmol phospholipid/ml), filtered (0.22 μm Nalgene filter), and thenpasses serially through 18, 25 and 30 gauge needles.

The size profile of each batch of liposomes was determined by afluorescent activated cell sorter (Coulter Epic 753 Dye Laser System,Coulter Electronics, Hyaleah, Fla.) from linear and logarithmic forwardangle light scattered signals at 488 nm at 1000 mwatts. Latex beads(0.1, 0.22 and 0.51 μ in diameter) were used as size markers andapproximately 20,000 signals were acquired per measurement. Since thecharge, size and structure of L-proline is similar to that of cHyp,encapsulation efficiency of cHyp was estimated from the percententrapment of 10 μCi of [¹⁴ C]-L-proline into the liposome pelletfollowing centrifugation.

EXAMPLE 16 LIPOSOME ENCAPSULATED cHYP ADMINISTRATION

In this example, the liposome encapsulated antifibrotic agent of theinvention was tested and compared with alternative formulations andmodes of administration of the same antifibrotic agent. Accordingly, theproline analogue cis-4-hydroxy-L-proline (cHyp) entrapped in liposomeswas administered to rats developing hypoxic pulmonary hypertension.

METHODS

Materials

Materials were L-α-dipalmitoylphosphatidylcholine (780 g/mol) (AvantiPolar Lipids, Birmingham, Ala.), cholesterol (386.6 g/mol) andstearylamine (269.5 g/mol) (Sigma Chemical Co., St. Louis, Mo.),cis-4-hydroxy-L-proline (cHyp) (Calbiochem Corp., La Jolla, Calf.), [¹⁴C]-L-proline (260 mCi/mM) and methanol and quaternary ammonium hydroxide(Protosol, New England Nuclear Co., Boston, Mass.), fluorescent latexmicrospheres (Fluoresbrite, Polysciences, Inc., Warrington, Pa.), 1,1',dioctadecyl-3,3,3', 3'-tetramethylindorbocyanine perchlorate (D282,Molecular Probes, Inc., Eugene, Oreg.), and rabbit anti-factor VIIIantibody and FITC goat-anti-rabbit antibody (Calbiochem Corp., La Jolla,Calf.). Chemicals were analytical grade.

Animals

Six week old male Sprague Dawley rats (Crl:CD[SD]BR) weighing 185-205 gand 8 week old female Swiss mice (Crl:CP-1[ICR]BR) weighing 30-32 g(Charles River Breading Laboratories, Wilmington, Mass.) were maintainedin a holding area one week prior to study and were fed food and water adlibitum. Rats were randomly allocated to hypoxia or air groups; micebreathed air. Animals were kept in a 12-hour light-dark cycle.

Exposure Conditions

Four rats were placed in a polycarbonate chamber measuring 51×41×22 cm,and humidified gas (10% O₂, 90% N₂ flowed into the chamber at a rate of400 ml/min. Gas samples were analyzed electrometrically (model MB53MK2,Radiometer, Copenhagen, Denmark); PO₂ ranged from 74-80 mmHg and PCO₂from 3-5 mmHg. Air-breathing rats were kept in cages in the same roomand were pair-fed to hypoxic animals by weighing the food consumed byhypoxic animals and feeding the same amount of food to air-breathinganimals to ensure that final body weights were similar. The chamberswere opened once daily for 10 min. to clean, weigh and feed the animals.

Hemodynamic Measurements and Heart Weight

A catheter was placed in the right ventricle of anesthetized rats (50mg/kg pentobarbital intraperitoneally), and mean right ventricularpressure was measured using a pressure transducer (model P23Db, Statham,Instruments, Oxnard, Calf.) and recorded (model SP-2006, StathamInstruments). Pressure was measured after the animal had breathed airfor 20 min. to eliminate the tonic response to hypoxia. After sacrificeby abdominal aorta transection, hematocrit and ratio of ventricularweights were measured, and the position of the catheter was confirmed atautopsy.

Biochemistry

Main pulmonary artery (9 mm in length) was excised and analyzed fortotal protein and hydroxyproline contents as previously described.Tissue was hydrolyzed in 6N HCl at 118° C. for 48 hrs., diluted 1:10 inwater, and a 0.1 ml aliquot was assayed for total protein by theninhydrin method using leucine as standard and for hydroxyproline by acolorimetric method. Results of triplicate measurements were expressedas content per vessel.

Preparation of Liposome

Unilamellar, positively charged phospholipid vesicles (liposomes) wereprepared by reverse phase evaporation as previously described, exceptthat the lecithin component was replaced with 97.5 mgL-α-dipalmitoylphosphatidylcholine.

Characterization of Liposomes

Liposome diameter was estimated by a single beam fluorescent activatedcell sorter (Epic 752 Dye Laser System, Coulter Electronics, Hialeah,Fla.) using an argon ion laser emitting a 488 nm (1 watt). Latexmicrospheres (0.10-0.51 μm diameter) were used as size markers.Liposomes or microspheres were suspended in PBS, and size histogramswere analyzed using a computer system (Easy 88 Epinet, CoulterElectronics) interfaced with the fluorescent activated cell sorter. Thediameter of 90% of the liposomes ranged between 0.10 to 0.22 μm.Entrapment efficiency of cHyp into liposomes was estimated bysubstituting 10 μCi [¹⁴ C]-L-proline in place of cHyp (see above). A 0.1ml aliquot of the [¹⁴ C]-L-proline entrapped liposome was added to 5 mlscintillation fluid (Liquiscint, National Diagnostics, Somerville, N.J.)and counted at 94% efficiency using a liquid scintillation counter(Tri-Carb, Packard Instruments, Downers Grove, Ill.). Percentencapsulation was estimated as the percentage of counts in liposomes andwas found to be 51±6% (n=11) and remained constant during storage at 4°C. for 21 days.

Injections

Cis-4-hydroxy-L-proline dissolved in saline (free cHyp) or saline alonewere injected subcutaneously (0.5 ml) or intravenously. Intravenousinjections were performed in anesthetized animals (25 mg/kg thiopental,intraperitoneally). In rats, liposomes containing cHyp or emptyliposomes were injected intravenously (18 μmol phospholipid in -0.5 ml)via the dorsal vein of the penis over 5 sec using a 30 gauge needle. Inmice, liposomes (18 μmol phospholipid in 0.5 ml) were injected into thetail vein.

Mode of Delivery and Dose of cHyp

Four modes of delivery of cHyp were used in rats. Free cHyp (200 or 100mg/kg) was injected subcutaneously twice daily during exposure tohypoxia. A single dose of free cHyp (200 mg/kg) was given intravenouslyprior to exposure to hypoxia.

Single doses of cHyp entrapped in liposomes (200 or 100 mg/kg) wereinjected intravenously prior to exposure to hypoxia.

Multiple doses of cHyp in liposomes (200 mg/kg) were injectedintravenously prior to hypoxia and every 5 days during exposure tohypoxia.

Single doses of cHyp entrapped in liposomes after reticuloendothelialblockage were produced by intravenous injection of a single dose ofempty liposomes followed 30 minutes later by a single intravenous doseof cHyp entrapped in liposomes (100 or 50 mg/kg) prior to exposure tohypoxia.

The purpose was to enhance the localization of liposomes containing cHypto the lungs by prior treatment with empty liposomes as temporaryreticuloendothelial blocking agents.

General Protocol

In each animal, we assessed the effect of injection of cHyp on fiveparameters of exposure to hypoxia: mean right ventricular pressure (RVP)measured after the animal had been removed from the hypoxic environment,ratio of ventricular weights (RV/[LV+S]), hematocrit, and the contentsof hydroxyproline and protein in the pulmonary artery. For eachexperimental group, comparisons were made to a group exposed to hypoxiaand injected with a control substance and to a group exposed to air. Forfree cHyp, the control substance was saline; for cHyp entrapped inliposomes, the control substance was empty liposomes. Groups wereage-matched; the air group was weight-matched to the hypoxic groupinjected with the control substance. Average results of each parameterwere compared.

Experimental Protocols

Twelve groups of rats were exposed to hypoxia and injected with cHyp(Groups 1-12, Table 1). Three groups were exposed to air and injectedwith cHyp (Groups 13-15, Table 1).

Groups were used to compare the mode of delivery of cHyp, various dosesusing the same mode of delivery, and the duration of effect of single ormultiple injections of cHyp. Six experimental protocols were used.

The first protocol studied whether cHyp delivered in liposomes was moreeffective than free cHyp in preventing pulmonary hypertension. Efficacyfor each mode of drug delivery was determined as the minimal dose ofcHyp required to prevent pulmonary hypertension after 3 days exposure tohypoxia. Free cHyp was given as 200 or 100 mg/kg subcutaneously twicedaily (Groups 1 and 2). Free cHyp was also given as a single dose of 200mg/kg intravenously prior to hypoxia (Group 3). Groups 1-3 were comparedto groups exposed to air and hypoxia for 3 days and injectedsubcutaneously twice daily with saline. Groups 1 and 2 were alsocompared to groups given liposome-entrapped cHyp as a single intravenousinjection of 200 or 100 mg/kg prior to exposure to hypoxia (Groups 4 and5). Groups 4 and 5 were compared to a group exposed to hypoxia for 3days and given a single intravenous injection of empty liposomes priorto hypoxia.

The second protocol studied the duration of antihypertensive effect of asingle dose of 200 mg/kg cHyp entrapped in liposomes injected prior toexposure to hypoxia. Groups were studied after 3, 5 or 7 days ofexposure to hypoxia (Groups 4, 6 and 7). Results were compared toage-matched air groups and groups injected with single doses of emptyliposomes after 3, 5 or 7 days exposure to hypoxia.

The third protocol studied whether 200 mg/kg cHyp entrapped in liposomeinjected intravenously prior to and every 5 days during exposure tohypoxia prevented pulmonary hypertension on day 21 (Group 8). Resultswere compared to a group exposed to air for 21 days and to a groupinjected with empty liposomes prior to and every 5 days during a 21-dayexposure to hypoxia.

The fourth protocol studied whether reticuloendothelial blockade priorto injection of cHyp entrapped in liposomes improved drug action.Reticuloendothelial blockade was produced by a single intravenousinjection of empty liposomes (18 μmol phospholipid in 0.5 ml) 30 min.prior to the injection of cHyp in liposomes. Groups given 100 or 50mg/kg cHyp intravenously after reticuloendothelial blockade (Groups 9and 10) were compared to an air group and to a hypoxic group injectedwith 100 mg/kg cHyp without reticuloendothelial blockade (Group 5).Groups were compared at 3 days after exposure to hypoxia.

The fifth protocol compared the duration of effect of a single dose of100 mg/kg cHyp entrapped in liposomes after reticuloendothelial blockadeand studied at 3, 5 and 7 days of hypoxia (Groups 9, 11 and 12). Resultswere compared to an air group and to groups with reticuloendothelialblockade injected with single doses of empty liposomes and studied ondays 3, 5 and 7 of hypoxia.

The sixth protocol studied whether cHyp injected in air breathing ratsaffected any of the parameters of exposure to hypoxia. Air groups weregiven free cHyp 200 or 100 mg/kg subcutaneously twice daily for 3 days(Groups 13 and 14) and were compared to saline injected animals. A groupwas injected with 200 mg/kg of cHyp in liposomes every 5 days during a21-day exposure to air (Group 15), and results were compared to a groupinjected with empty liposomes every 5 days during a 21-day exposure toair.

Effect of Acute Injection of Liposomes on Right Ventricular Pressure

One group of anesthetized, catheterized, air-breathing rats was injectedwith a bolus of liposomes to determine the acute pressor effect ofliposomes. After RVP was stable for 5-10 min., a bolus of emptyliposomes (18 μmol phospholipid in 0.5 ml) was injected via the dorsalvein of the penis, and blood pressure was recorded continuously until itreturned to baseline. The maximal increase in RVP during the first 2min. after injection was compared to the blood pressure during theperiod prior to injection.

Uptake of Radiolabelled Liposomes by Pulmonary Artery Endothelial Cellsin Culture

Fresh bovine pulmonary arteries were perfused with sterile PBScontaining 0.1 mg/ml gentamicin, 37° C., until free of blood. Theendothelial cells were mechanically removed and placed in Medium 199containing 10% fetal bovine serum, 5% calf serum, IU/ml penicillin, 100μg/ml streptomycin, and 0.05 mg/ml gentamicin, pH 7.4, and not fed ormoved for at least one week. Thereafter, dividing cultures were fedtwice weekly and passaged 7 times using a 2:1 split. Endothelial cellswere identified by their characteristic cobblestone appearance inculture and the presence of angiotensin converting enzyme and factorVIII-related antigen by immunofluorescence. Endothelial cells (1×10⁵)and 100 μl of the above medium were added to each 38 mm² well of a96-well flat bottom plate (Microtest II, Falcon Plastics, Oxnard,Calf.). Aliquots of liposomes containing [¹⁴ C]-L-proline (0.1 μCi, 0.2μmol phospholipid, 5 μl per well) were added to the cultured cells.Separate wells were used to measure uptake at intervals from 30 min. to5 hr. After incubation, cells were washed 3 times with PBS, removed with0.1 M sodium hydroxide, and radioactivity in a 500 μl aliquot counted ina liquid scintillation counter. The percent uptake of liposomes wasestimated as the percentage of total radioactivity added per well.

Localization of Fluorescent Dye Entrapped in Liposomes in PulmonaryArtery Endothelial Cells in Culture

To study whether liposomes are taken up by endothelial cells, liposomes(0.8 μmol phospholipid, 20 μl per well) containing the lipophilicfluorescent dye D282 were added to endothelial cells in culture for 0,30 min., 1, 2, 3 and 5 hr. The cells were washed three times with mediumand viewed using a microscope equipped with a fluorescence attachment.Endothelial cells with addition of empty liposomes were evaluated forautofluorescence.

Organ Distribution of Radiolabelled Liposomes

We estimated the distribution and retention of liposomes in selectedorgans by injecting radiolabelled liposomes in air-breathing mice andmeasuring radioactivity in the organs at times after injection. Micewere injected with [¹⁴ C]-L-proline in liposomes (2.2×10⁵ dpm in 100 μl)over one sec via the tail vein using a 30 g needle. Animals were killedby cervical dislocation at 1, 2, 6, 24, 48 and 72 hr. after injection.The lungs, heart, liver, spleen and kidneys were removed, rinsed insaline, blotted dry and weighed. A portion of each organ (100 mg) wassolubilized in 2 ml methanol and quaternary ammonium hydroxide for 24 hrat 60° C. in a shaking water bath. A 100 μl aliquot of the suspensionwas added to 5 ml scintillation fluid (Econofluor, New England NuclearCo., Boston, Mass.) and 2 ml methanol and quaternary ammonium hydroxideand counted in triplicate in a liquid scintillation counter. Counts werecorrected for quenching by each tissue, and results were expressed aspercent of total injected dose in each organ.

Statistical Analysis

Mean ± SEM from each group were obtained. Data were analyzed by one-wayANOVA followed by Duncan's post-hoc test. Non-parametric data (animalsurvival) were analyzed by a continuity adjusted Chi-square analysiswith Yates' correction. A P value of 0.05 was considered significant.

RESULTS

In General

Substantially lower doses of cHyp were effective in preventing pulmonaryhypertension and collagen accumulation in pulmonary arteries when givenintravenously in liposomes compared to subcutaneous administration ofthe free agent. Moreover, a single intravenous dose of cHyp entrapped inliposomes had a sustained effect on suppressing pulmonary hypertension.Delivery of an antifibrotic agent in liposomes improves drug action inthe treatment of experimental pulmonary hypertension.

Animals

Survival was 128 of 130 (98%) in combined air groups and 165 of 192(86%) in the combined hypoxic groups (X² - 5.8, P<0.05). Survival at 3days of animals exposed to hypoxia and injected with saline or free cHypwas 13 of 16 (81%); survival of animals exposed to hypoxia and injectedwith liposomes was 51 of 61 (84%) (NS). After 3 days, 14 deaths occurredin the hypoxic group treated with liposomes (there were no age-matchedsaline or free cHyp treated animals exposed to hypoxia to comparesurvival). Initial body weight was 198±4 g (mean±SEM) (n=322); finalbody weights were: day 3, 190-202 g; day 5, 204-208 g; day 7, 202-208 g;and day 21, 225-230 g. We found no differences on any day in final bodyweights among hypoxic animals treated with cHyp, hypoxic animals treatedwith the test substance and air-breathing animals.

Hypoxia and Treatment with Empty Liposomes

Exposure to hypoxia from day 0 to day 21 produced progressive increasesin all parameters in rats injected with empty liposomes; RVP increasedfrom 9±1 to 21±2 mmHg, RV/(LV+S) from 0.24±0.01 to 0.43±0.02, hematocritfrom 48±1 to 66±1%, hydroxyproline content from 74±4 to 163±14 μg/vesseland protein content from 1.2±0.1 to 3.2±0.3 mg/vessel (n=7-8, allP<0.05). All parameters were increased as early as 3 days exposure tohypoxia (Table 2).

Free vs. Liposome-Entrapped cHyp

The effect of free cHyp on preventing pulmonary hypertension at 3 daysis shown in Table 2. Treatment with 200 mg/kg cHyp subcutaneously twicedaily for 3 days produced reductions in all 5 parameters compared to thesaline injected hypoxic group. However, the values were greater thanthose in the air group, indicating that free cHyp partially preventedpulmonary hypertension. Injection of 100 mg/kg free cHyp subcutaneouslyfor 3 days did not prevent increased RVP, RV/(LV+S) or hydroxyproline orprotein contents; there was partial decrease in hematocrit (Table 2).Free cHyp injected intravenously prior to hypoxic exposure had no effecton any parameter (Table 2). A single dose of 200 mg/kg cHyp entrapped inliposomes injected prior to exposure to hypoxia partially preventedincreases in RVP and hematocrit and completely prevented increases inRV/(LV+S) and contents of hydroxyproline and protein in pulmonaryarteries at 3 days (FIG. 1). A single intravenous dose of 100 mg/kg cHypentrapped in liposomes had no protective effect on any of the parametersat 3 days.

Duration of a Single Dose of cHyp Entrapped in Liposomes

A single intravenous injection of 200 mg/kg cHyp prior to exposure tohypoxia partially or completely prevented increases in RVP, RV/(LV+S)and hydroxyproline content of pulmonary artery at 3 and 5 days;increases in hematocrit and protein content were prevented at 3 days butnot at 5 days (FIG. 1). At 7 days, a single injection of 200 mg/kg cHypin liposomes did not prevent increases in any of the measured parameters(FIG. 1). Thus, a single intravenous injection of 200 mg/kg cHyp inliposomes prior to exposure to hypoxia partially suppressed thedevelopment of pulmonary hypertension, right ventricular hypertrophy andpulmonary artery collagen accumulation for 5 days.

Intermittent Doses of cHyp Entrapped in Liposomes

Intermittent injections of cHyp in liposomes every 5 days during the 21day exposure period partially prevented the increases in RVP, RV/(LV+S)and hydroxyproline and protein contents of pulmonary artery; there wasno effect on hematocrit (Table 3). These results show that intermittentdoses of single doses of cHyp in liposomes suppress the development ofpulmonary hypertension for as long as three weeks.

Single Dose of cHyp Entrapped in Liposomes after ReticuloendothelialBlockade

In animals with reticuloendothelial blockade, a single dose of 100 mg/kgcHyp entrapped in liposomes partially prevented the increases in RVP,RV/(LV+S) and hydroxyproline content of the pulmonary artery at 3 days;there was no apparent effect on hematocrit and protein content ofpulmonary artery at 3 days (FIG. 2). A dose of 50 mg/kg had noprotective effect on any parameter at 3 days (FIG. 3). Since the minimaleffective dose of cHyp in liposomes without reticuloendothelial blockadewas 200 mg/kg, these results suggest that reticuloendothelial blockadeprior to a single dose of cHyp in liposomes results in a lower effectivedose of cHyp.

Duration of a Single Dose of cHyp Entrapped in Liposomes afterReticuloendothelial Blockade

In animals with reticuloendothelial blockade, treatment with a singleintravenous injection of 100 mg/kg prior to exposure to hypoxiapartially or completely prevented increases in RVP, RV/(LV+S) andhydroxyproline content of the pulmonary artery at 3 and 5 days; therewas no effect on hematocrit or protein content (FIG. 2). At 7 days aftera single injection, the agent did not prevent increases in any of themeasured parameters (FIG. 2). The pattern of suppression was similar tothat found without reticuloendothelial blockade (FIG. 1), except thedose was 100 mg/kg instead of 200 mg/kg.

CHyp in Air-Breathing Rats

There was no effect of 200 mg/kg or 100 mg/kg cHyp injected twice dailysubcutaneously for 3 days on any of the measured parameters (Table 2).Also, intermittent intravenous injections of 200 mg/kg cHyp in liposomesevery 5 days during a 21-day air exposure period had no effect on anyparameter (Table 3).

Effect of Injection of Liposomes on Right Ventricular Pressure

Mean right ventricular pressure increased from 9±1 to 11±1 (mmHg) (n=5)within 2 min after injection of cHyp entrapped in liposomes (P<0.05).Injection of saline under the same conditions had no effect on RVP (9±1vs. 10±1 mmHg, n=4).

Uptake of Liposomes by Endothelial Cell in Culture

Percent uptake of liposome containing [¹⁴ C]-L-proline by pulmonaryartery endothelial cells was 3.3±0.3% at 30 min. Uptake was maximal at5.4±0.3% after 2 hr and remained at that level for 5 hr (FIG. 3).

Localization of Fluorescent Dye Entrapped in Liposomes

At 30 min after incubation with the fluorescent dye D282, a diffusepattern of immunofluorescence was observed in endothelial cell membranes(FIG. 4). At 2 hr a few fluorescent intracellular vesicles appearedwhich became more abundant at 3 to 5 hr after incubation.Autofluorescence was absent in cells not incubated with D282.

Organ Distribution of Radiolabelled Liposomes

Soon after administration of [¹⁴ C]-L-proline entrapped in liposomes,radioactivity appeared in the lung where it reached a maximum of 49±14%of total injected dose during the first 20 min (FIG. 5). There was arapid decrease in lung activity reaching a value of 5±1% at 6 hr. Spleentook up a greater proportion of radioactivity (9±1% at 6 hr) andretained -7-9% for up to 72 hr. Liver retained about the same amount aslung; heart and kidney contained <2% activity after 6 hr (not shown).After 72 hr, the lung contained 5±1% of total activity (FIG. 5).

DISCUSSION

The examples above demonstrate that the intravenous injection of cHyp inliposomes partially prevents the development of pulmonary hypertensionin rats exposed to hypoxia. Liposome entrapment was necessary for drugaction since intravenous injection of free cHyp was ineffective.

Compared to subcutaneous administration, intravenous delivery of cHyp inliposomes required considerably less total dose of drug to preventhypertension. Moreover, delivery of cHyp in liposomes resulted insustained drug effect; pulmonary hypertension was suppressed for 5 daysafter a single intravenous injection of cHyp in liposomes. This effectcould be extended for as long as three weeks by a single injection every5 days. Drug action on the pulmonary circulation could be improved byblocking uptake by reticuloendothelial organs prior to delivering theagent.

cHyp was chosen to test the effect of liposome delivery of drugs toblood vessels because it consistently prevents the early hemodynamic andbiochemical changes of hypoxic pulmonary hypertension in the rat. Theminimal total dose of cHyp required to prevent hypoxic pulmonaryhypertension using different modes of delivery was compared.

At 5 days the results were as follows: subcutaneous, 400 mg/kg (200mg/kg twice daily); single dose entrapped in liposomes withoutreticuloendothelial blockade, 200 mg/kg; single dose entrapped inliposomes with reticuloendothelial blockade, 100 mg/kg. Over the 5-dayinterval, the dose using cHyp entrapped in liposomes followingreticuloendothelial blockade was approximately 20 times more effectivethan a subcutaneous dose of free cHyp.

The assumption is made that cHyp is released from liposomes in thevicinity of vascular cells synthesizing collagen, thereby preventingaccumulation of collagen. There are two general pathways which liposomesmight follow to enter the blood vessel wall. First, liposomes injectedintravenously may be taken up by pulmonary vascular endothelial cells.Liposomes pass easily into reticuloendothelial organs because theendothelium of these organs is fenestrated. In organs with tightendothelium, such as lung, liposomes remain associated with endothelialsurfaces until they are degraded or endocytosed. Although it was shownthat liposomes are taken up by endothelial cells in vitro, there is noevidence that this process occurred in vivo. Second, liposomes may betaken up by circulating blood phagocytes and migrate into the lungtissue. Blood monocytes phagocytose liposomes and subsequently migrateto the alveoli to become alveolar macrophages. The analogue may bereleased from blood cells as they pass through the blood vessel walls.Liposomes are also phagocytosed by pulmonary intravascular macrophages,but the rat has few if any of these cells. Either of these two pathwaysmay be involved in release of cHyp to blood vessel walls.

These biochemical mechanisms probably account for the decreasedaccumulation of collagen in pulmonary arteries in the Examples. Inaddition, collagen synthesis in main pulmonary arteries of rats ismarkedly increased within 3 days of exposure to hypoxia and remainselevated for 7 days. Collagen synthesis is increased only in thepulmonary artery, probably because hypoxia causes structural remodelingin response to hypoxic hypertension in the pulmonary circulation.Proline analogues impair collagen formation in tissues undergoingincreased collagen synthesis, such as the pulmonary artery in earlyhypoxic pulmonary hypertension.

Treatment with cHyp is relatively specific for inhibiting collagensynthesis. For example, doses of cHyp which inhibit collagenaccumulation do not affect elastin accumulation. Nevertheless, it wasobserved that treatment with cHyp prevented increases in total proteinaccumulation at 3 days. Suppression of protein accumulation cannot beaccounted for by the decreased collagen since collagen synthesiscontributes only about 4-5% of the total protein synthesis inhypertensive pulmonary arteries of rats. One explanation is that cHypmay have interfered with the ability of vascular smooth muscle cells andfibroblasts to proliferate, since cHyp inhibits proliferation ofcultured cells by blocking collagen secretion required for cells toattach and grow. Marked cell proliferation occurs in hilar pulmonaryarteries of rats 2-3 days after onset of hypoxia, and suppression ofthis proliferation by cHyp may explain why protein content wassuppressed is early hypertension. At 5 days and later, there is littlecell proliferation and cHyp has no effect on protein accumulation after3 days.

Hypoxia-induced polycythemia was inhibited by the higher doses of cHyp,an effect previously noted with subcutaneous injection of cHyp.

The rate of rise of right ventricular pressure and hydroxyprolinecontent were similar between 0 and 3 days in the hypoxic group andbetween 5 and 7 days in the hypoxic groups given a single injection ofcHyp in liposomes (FIGS. 1 and 2). It was speculated that thesuppressive effect of cHyp in liposomes diminished after 5 days, andcollagen accumulated rapidly in the blood vessel wall. The late increasein collagen may have contributed to narrowing of the vascular lumenproducing hypertension. The stimulus for rapid collagen accumulation atflow-resistive sites, presumably hypoxic vasoconstriction, persistedduring hypoxia, but the added effect of collagen accumulation occurredonly after the drug effect wore off.

It is possible to enhance the delivery of liposomes to the lung byblockade of the reticuloendothelial organs. With blockade, half as muchcHyp is required in liposomes to prevent the rise in pulmonary bloodpressure as without RES blockade, suggesting that blockade produced ashift toward a greater portion of injected liposomes to the lung.

Intravenously injected liposomes will be initially distributed to lungssince it is the first organ they contact. Thereafter, liposomes aredistributed to other organs or are excreted. A small fraction of thetotal radioactivity (4-5%) remains in the lung for as long as 3 days,incorporated into tissue protein or retained in liposomes. Thesefindings are consistent with the observation that cHyp within the lungis available to inhibit collagen accumulation for up to 5 days afterintravenous injection.

In conclusion, the results show that intravenous injection of an agententrapped in liposomes substantially improves the action of an agentwhich inhibits the development of hypertension, probably by delivering alocally high concentration of drug which is released over time withinthe blood vessel wall. The encapsulated agent given intravenously wasapproximately 20 times more effective than the unencapsulated agentgiven subcutaneously.

                                      TABLE 3                                     __________________________________________________________________________    EXPERIMENTAL GROUPS                                                           Mode of Delivery          Frequency of Injection, Exposure                                                                  Duration of                     of cHyp     Route Dose (mg/kg)                                                                          Hypoxia             Exposure (days)                                                                       Group                   __________________________________________________________________________                                                          Number                  Free        sc    200     Twice daily during hypoxia                                                                        3       1                                   sc    100     Twice daily during hypoxia                                                                        3       2                                   iv    200     Single dose prior to hypoxia                                                                      3       3                       Lipsomes, single doses                                                                    iv    200     Single dose prior to hypoxia                                                                      3       4                                   iv    100     Single dose prior to hypoxia                                                                      3       5                                   iv    200     Single dose prior to hypoxia                                                                      5       6                                   iv    200     Single dose prior to hypoxia                                                                      7       7                       Liposomes, intermittent                                                                   iv    200     Prior to hypoxia & every 5 days                                                                   21 ing  8                       doses                     hypoxia                                             Lipsomes, single doses,                                                                   iv    100     Empty liposomes followed 30' later                                                                3y      9                       reticuloendothelial       single dose prior to hypoxia                        blockage           50                         3       10                                  iv    100                         5       11                                  iv    100                         7       12                                                Exposure to Air                                     Free        sc    200     Exposure twice daily during air                                                                   3       13                                  sc    100                         3       14                      Liposomes, single doses                                                                   iv    200     Every 5 days during exposure                                                                      21      15                      __________________________________________________________________________     Abbreviation: cHyp, cis4-hydroxy-L-proline; free cHyp not contained in        lipsomes; lipsomes, cHyp entrapped in liposomes; sc, subcutaneous; iv,        intravenous                                                              

                                      TABLE 4                                     __________________________________________________________________________    EFFECTS OF INJECTION OF FREE cHyp ON HEMODYNAMIC                              AND BIOCHEMICAL MEASUREMENTS ON 3 DAYS                                                      RVP  RV/(LV + S)                                                                           Hct  Hydroxyproline                                                                        Protein                               Exposure/Regimen                                                                         n  (mmHg)                                                                             (%)     (%)  (mg/vessel)                                                                           (mg/vessel)                           __________________________________________________________________________    Air, Saline                                                                              6   9 ± 1                                                                          0.24 ± 0.01                                                                        48 ± 1                                                                          75 ± 4                                                                             1.2 ± 0.1                          Hypoxia, saline                                                                          8  14 ± 1*                                                                         0.30 ± 0.01                                                                        54 ± 1*                                                                         90 ± 2*                                                                            1.7 ± 0.1*                         Hypoxia, free cHyp                                                                       10 10 ± 1**                                                                        0.25 ± 0.01**                                                                      51 ± 1**                                                                        78 ± 4**                                                                           1.4 ± 0.1**                        200 mg/kg sc bid × 3                                                    days                                                                          100 mg/kg sc bid ×                                                                 5  14 ± 1                                                                          0.31 ± 0.02                                                                        51 ± 1**                                                                        94 ± 4                                                                             2.3 ± 0.3                          3 days                                                                        200 mg/kg iv × 1                                                                   9  14 ± 1*                                                                         0.32 ± 0.01*                                                                       55 ± 1*                                                                         88 ± 7*                                                                            2.3 ± 0.3*                         injection                                                                     Air, free cHyp                                                                200 mg/kg sc bid ×                                                                 6    9 ± 1                                                                         0.24 ± 0.01                                                                        46 ± 1                                                                          72 ± 5                                                                             1.2 ± 0.1                          3 days                                                                        100 mg/kg sc bid ×                                                                 4   9 ± 1                                                                          0.24 ± 0.01                                                                        47 ± 1                                                                          70 ± 2                                                                             1.2 ± 0.1                          3 days                                                                        __________________________________________________________________________     Values, mean ± SEM. Measurements taken 3 days after exposure to air on     10% O.sub.2. n = number animals/group; cHyp, cis4-hydroxy-L-proline; iv,      intravenous; sc, subcutaneous; bid, twice daily; RVP, mean right              ventricular pressure; RV/LV + S), ratio of ventricular weights; Hct,          hematocrit; *, P,, 0.05 compared with hypoxia                            

                                      TABLE 5                                     __________________________________________________________________________    EFFECTS OF INTERMITTENT INJECTIONS OF cHyp IN LIPOSOMES                       ON HEMODYNAMIC AND BIOCHEMICAL MEASUREMENTS ON DAY 21                                                            Hydroxyproline                             Exposure/Regimen                                                                        n RVP (mmHg)                                                                            RV/(LV + S) (%)                                                                         Hct (%)                                                                            (mg/vessel)                                                                           Protein (mg/vessel)                __________________________________________________________________________    Air, empty liposome                                                                     8  9 ± 1                                                                             0.24 ± 0.01                                                                          47 ±                                                                             79 ± 6                                                                            1.5 ± 0.1                       Air, liposomes,                                                                         8  9 ± 1*                                                                            0.24 ± 0.01                                                                          46 ± 1                                                                           84 ± 3                                                                            1.4 ± 0.2                       cHyp                                                                          Hypoxia, empty                                                                          7 21 ± 2                                                                             0.43 ± 0.02*                                                                         66 ± 1**                                                                        163 ± 14*                                                                          3.2 ± 0.3*                      Hypoxia, lipsomes,                                                                      7 15 ± 1**                                                                           0.36 ± 0.01**                                                                        68 ± 1*                                                                         121 ± 12**                                                                         2.4 ± 0.2**                     cHypo                                                                         __________________________________________________________________________     Values, mean ± SEM. Measurements taken 3 days after exposure to air on     10% O.sub.2. n = number animals/group; cHyp, cis4-hydroxy-l-proline; bid,     twice daily; RVP, mean right ventricular pressure; RV/LV + S), ratio of       ventricular weights; Hct, hematocrit; *, P,,0.05 compared with hypoxia   

Any of the antifibrotic agents other than cHyp can also be liposomeencapsulated and administered to treat fibrotic conditions. Each of theantifibrotic agents can be administered in liposomes in an amounteffective to treat diseases where collagen metabolism is of concern.

The antifibrotic agents can also be linked to a monomer and incorporatedinto lipsomes. For purposes of illustration, the antifibrotic agentbelow is cHyp linked to ethylene glycol.

    cHyp--OCH.sub.2 CH.sub.2 --OH

The linkage could again be an ether, ester or another linkage. Also, anadditional antifibrotic compound can be linked to the glycol through thehydroxyl group.

If ethylene glycol is used as the monomer, safety and toxicity may needto be taken into account. A preferred monomer in this regard would bepropylene glycol or another suitably non-toxic monomer.

A polymeric form which can also be included herein is the polymer:

cHyp-PEG or

cHyp-(PEG-cHyp)_(y)

The cHyp can be linked directly or through a linking compound. Also, thecHyp can be substituted in whole or in part with another antifibroticagent. The variable Y in this case can be an integer from 1 up to about100.

As can be noted with respect to FIGS. 6 through 9, the antifibroticagents can be conjugated with PEG or another polymer and used to reducecellular proliferation in the presence collagen metabolism. In FIG. 6,the effect of free cHyp, polymeric cHyp and free trans hydroxyprolinewere compared over a six day period.

Smooth muscle cells were allowed to proliferate in the presence of freecHyp, polymeric cHyp and trans hydroxyproline. The polymeric cHyp wasproduced as described above and contained ester linkages. On each day,the cells were trypsinized and counted with a hemocytometer. Cellularproliferation was significantly reduced in the cHyp polymer group, ascompared to the free cHyp and tHyp groups. This is further supported bythe data represented in FIG. 9, generated with fibroblast cells.

When the polymer is conjugated with cHyp via amide linkages, cellularproliferation is further reduced. See, e.g., FIG. 7, which a comparisonis presented between the ester linked polymer and the amide linkedpolymer in FIG. 8.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A compound comprised of the reaction product ofan antifibrotic agent selected from the group consisting ofcis-4-hydroxy-L-proline, 3,4-dehydro-L-proline, cis-4-fluoro-L-proline,cis-4-chloro-L-proline, laevo and cis isomers of compounds of thegeneral structural formala: ##STR6## wherein R is Oh, Cl, F, NH₂, SH,SCH₃, OCH₃, ONO₂, OSO₂, OSO₃ H, and pharmaceutically acceptable saltsthereof, covalently linked to a low molecular weight polymer selectedfrom the group consisting of poly(ethylene glycol), poly(propyleneglycol), poly(isopropylene glycol), poly(butylene glycol) andpoly(isobutylene glycol) wherein said antifibrotic agent is covalentlybonded through a reactive group or linking compound to said lowmolecular weight compound.
 2. A compound in accordance with claim 2further comprised of a linking compound, which upon reaction covalentlylinks the antifibrotic agent and the low molecular weight polymer.
 3. Acompound in accordance with claim 2 wherein the linking compound isselected from the group consisting of amino acids and short chainpeptides comprised of up to five amino acid residues, ethanolamine andsuccinic acid.
 4. A compound in accordance with claim 2 wherein theantifibrotic agent is cis-hydroxyproline, the linking compound is lysineand the low molecular weight polymer is poly(ethylene glycol).
 5. Acompound of the formula: ##STR7## wherein PEG consists of a lowmolecular weight polymer of poly(ethylene glycol).
 6. A pharmaceuticalcomposition comprising a compound which is the reaction product of (a)an antifibrotic agent selected from the group consisting ofcis-4-hydroxy-L-proline; 3,4-dehydro-L-proline; cis-4-fluoro-L-proline;cis-4-fluoro-L-proline; cis-4-chloro-L-proline, laevo and cis isomers ofcompounds of the general structural formala: ##STR8## wherein R is OH,Cl, F, NH₂, SH, SCH₃ OCH₃, ONO₂, OSO₂, OSO₃ H, H₂ PO₄, or COOH, andpharmaceutically acceptable salts thereof, and (b) a low molecularweight polymer selected from the group consisting of poly(ethyleneglycol), poly(propylene glycol), poly(butylene glycol), poly(isobutyleneglycol) and poly(isopropylene glycol).
 7. A pharmaceutical compositionin accordance with claim 6 further comprised of (c) in linking compoundselected from the group consisting of lysine, tyrosine, arginine andshort chain peptides containing up to about five amino acid residues. 8.A pharmaceutical composition in accordance with claim 6 wherein theantifibrotic agent is cis-hydroxyproline, the low molecular weightpolymer is poly(ethylene) gylcol and the linking compound is lysine.